1. Field of the Invention
The present invention relates to a confocal microscope used for the observation of a specimen, e.g., a biological specimen.
2. Related Background Art
Generally, conventional confocal microscopes have been so constructed that the light beam emitted from a light source is condensed on a specimen and the backward light from the specimen irradiated by the condensed light spot is selectively detected through a light shielding member with a pinhole arranged at a position conjugate to the specimen, thereby producing image information. Such light shielding member is composed of a thin sheet of metal or a thin film of metal evaporated on the surface of a transparent substrate to shield undesired components in the backward light and a pinhole is formed at the required location of the light shielding member.
The features of the confocal microscope reside in that when projecting the light beam emitted from the light source on the specimen and detecting the backward light from the specimen, such undesired light beams as the return beam from around the condensed light spot on the specimen, the return beam from the out-of-focus portions, etc., are shielded by the light shielding member with a pinhole and therefore any undesired blurred image is prevented from superposing on the periphery of an image of the scanning condensing point.
Generally, among the confocal microscopes, the scanning-type fluorescent confocal microscope or the confocal microscope in which the laser-beam from a light source unit is projected as an excitation light on a specimen and the fluorescence produced from the specimen due to the excitation light is detected, has an excellent resolution along the optical axis direction.
This is an important feature which is not possessed by the ordinary fluorescent microscopes and this is extremely useful in the fields of medical science and biology where cells, biological tissues, etc., must be observed stereoscopically. In addition, by reducing the size of the pinhole of the light shielding member, it is possible to ensure more improved resolution along the optical axis.
Then, with the scanning-type confocal microscope employing fluorescence, as in the case where the excitation light is ultraviolet light and the backward beam is a visible light, for example, the excitation light and the backward beam differ in wavelength from each other and thus there is the danger of the objective lens showing an axial chromatic aberration and an aberration of magnification.
While it is possible to solve, by the use, for example, of a corrector lens, the defocusing problem of the backward light on the light shielding plate, in the case where the objective optical system having an axial chromatic aberration is used, when the correction is provided in this way, if the light deflecting element of the deflecting optical system for specimen scanning purposes is not arranged at the pupil position, a shading phenomenon is caused in which only the central portion of the observation image becomes bright in rectangular form and the other portions become dark. If the degree of this shading is extremely high, the peripheral portion of the observation image becomes dark completely and it cannot be observed.
Also, where the objective optical system shows a chromatic aberration of magnification, the shading is caused in the similar manner. In this case, a condition is caused in which the peripheral portion of the visual field becomes dark in circular form irrespective of the arrangement of the light deflecting element, and moreover the periphery of the observation image becomes dark completely and cannot be observed as in the case of the axial chromatic aberration if the degree of the shading is extremely high.
For instance, in the case of the confocal microscope utilizing the fluorescent wavelength, the excitation light or the projection light and the fluorescence of the backward beam to be detected differ in wavelength from each other. This is true not only with the fluorescent microscope but also with the confocal microscope in which the backward light of different wavelengths other than the reflected light are directly detected.
Thus, where a lens of a large chromatic aberration of magnification is used in the objective optical system, if the backward light from a specimen is condensed through the objective optical system and the deflecting optical system at a position conjugate to the condensing point on the specimen, there is the danger of the position of the condensing point of the backward light deviating from the pinhole position on the light shielding member.
Thus, there are instances where, of the fluorescence emitted from the condensed light spot portion on the specimen, some components condensed on the positions deviating from the pinhole are intercepted by the light shielding member thus failing to reach the detection system. In such a case, a shading phenomenon is caused in which the light quantity of the fluorescence detected is decreased and the peripheral portion of the observation image becomes dark in circular form.
Such shading not only deteriorates the indication condition of the observation image but also gives rise to a problem in that the measurement accuracy is deteriorated extremely in cases where the amount of calcium ions or the like is measured by means of the emitted (detected) fluorescent wavelength.
Such shading due to the chromatic aberration of magnification cannot be overcome unless the chromatic aberration of magnification is satisfactorily corrected within the objective optical system or the pinhole diameter of the light shielding member is increased considerably.
However, the former is disadvantageous in that such correction is extremely difficult from the optical designing point of view and also the selection of a lens free of such chromatic aberration of magnification, if any, causes an enormous increase in the production cost. On the other hand, the latter causes an undesired result of ruining the important feature of the confocal microscope, i.e., the resolution along the optical axis direction.